(This story is the continuation/conclusion of the article beginninghere)

Keeping a full
plate

According to Corning's Ludwig, another question—aside from
the
animal-free discussion—that he hears a lot is, "How can I make 2 trillion
cells?"

"They never really talk about it, but there is always this
question of 'how can I make a trillion cells cheaply?' There is a lot of
emphasis now on cost and cost-containment," he says.

This question will only get louder as more and more projects
move from the research departments and into the
clinic, and the demands for
large quantities of cells increases. With standard polystyrene plates, Ludwig
explains, you can really only get stacks of
about 40 plates because of the need
for sufficient headspace between the plates to allow adequate gas exchange.

"If you reduce the headspace, you get poor oxygen transfer
and the cells end up starving," he says.

To accommodate the demand for tighter stacking and increased
throughput, Corning introduced the HyperStack format,
which relies on a
gas-permeable polystyrene. Thus, oxygen transfer occurs through the permeable
film allowing the company to create a very dense
stack.

"But even with something like HyperStack, you can really
only get about 2 billion cells
with a 120-layer stack, so customers are really
actively looking at bioreactors with microcarrier beads," he adds.

"As more cell-based therapeutics progress toward clinical
testing, the consistency, quality and reproducibility of large-
scale culture
systems become an imperative," said Robert Shaw, commercial director of EMD
Millipore's Stem Cell Initiative, in a May press release.

EMD Millipore was announcing its collaboration with
PharmaCell BV to optimize large-scale expansion and harvesting of HepaRG cells
using
Mobius CellReady disposable bioreactor technology. In support of the
European BALANCE project, the collaboration is aimed at the development of a
bioartificial liver.

"Customers tend to like the flexible manufacturing format
that a single-
use bioreactor gives them, so they can quickly change over
without doing steam-in-place processes," Ludwig says. "The beads we have are a
little more
differentiated—because Corning is really big on surface science—so
the advantage that we have in the stem cell area is that we have surface
treatments,
media and the vessels together, which makes it easier for the
customers looking for a transitional system."

Several posters at the ISSCR conference were dedicated to
the development of microcarrier technologies, and the challenges of
shifting
from a two-dimensional platform to the third dimension of suspended cultures.

"The biggest
thing around the microcarrier beads is that
they seed differently; you can't just put some beads in suspension with some
cells, you have to let them
settle," Ludwig adds. "Developing that kind of
protocol to get them to seed on the beads and expand correctly is a little bit
challenging."

As he explains further, it is not just about the surface
materials per se and their cell
adhesion properties.

"It's a much more dynamic environment," he says. "Whenever
you have
things in motion, there are lots of concerns about how the
microcarriers are interacting with the impellers, and making sure that the
oxygen level and
heat is maintained from top to bottom."

A transitional offering between plates and a full
bioreactor
environment may be in the offing, however, with the announcement in April of
data arising from a collaboration between UCB and TAP Biosystems. In
particular, UCB tested the performance of a variety of cell lines in the 15-mL
ambr
microscale bioreactor and found the results were representative of similar
experiments with 100 L bioreactors, and yet would allow researchers to test up
to 24 clones in parallel.

While the experiments were performed on protein-expressing
CHO cell
lines rather than stem cells, the results nonetheless open the
possibility of testing performance at a smaller, more experimental level before
scaling
up to production levels.

Aside from the growth vessels, the surface chemistry is also
important
to stem cell research, and this is one area where AMSBIO has invested
heavily.

Thus, AMSBIO has developed an extensive portfolio of
extracellular matrices (ECMs), but whereas many companies simply offer
the ECM
proteins such as laminins, AMSBIO has gone one step further.

Rather than just extracting
the protein for a recombinant
laminin from a cell line, the company has developed recombinant versions where
the cell-binding motifs of the ECM protein
are fused to a naturally adhesive
protein derived from mussels that makes it easier to stick the ECMs to plates.

"You can coat your plate with just one of these motifs, or
any combination of these motifs in any concentration," says
Pridham-Field. "The
idea is to grow your stem cells on these defined matrices and optimize the
environment."

One of the challenges for researchers, he adds, is knowing
which motifs to use for their specific experiments, a question that
AMSBIO does
not really have the capacity to answer.

"Because we recognize there is a limit as to
how much we can
tell people, we'll put together a 96-well plate of various motif combinations,"
he says, describing something akin to a chocolate
sampler box. "They can then
put the same cells in each well and see how they react."

As a
follow-through on its purchase of Discovery Labware,
Corning too
continues to develop a portfolio of peptide surface treatments.

"Discovery Labware had invested heavily
in peptide surface
treatments, so it really complimented Corning quite well," recounts Ludwig, but
where Corning had focused its efforts on letting
the customer coat the plates,
Discovery Labware focused on precoated cultureware, a practice that Corning has
largely adopted.

The battle continues to expand the repertoire of current
stem cell applications, while keeping an eye on the
downstream possibilities of
sending those same cells into the clinic as therapeutics—which means that as
researchers and companies do their best to
figure out how to make stem cell
resources less expensive and more effective, others will continue to search for
places to put all this stuff.